Journal: Cell
Article Title: A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells
doi: 10.1016/j.cell.2016.08.052
Figure Lengend Snippet: A) Gata3, a zinc-binding TF, ranks first in the dysfunction module. B and C) WT mice were implanted subcutaneously with B16F10 melanoma cells. TILs were isolated on day 15 and analyzed for Gata3 expression and T cell function. B) Representative flow cytometry data showing Gata3 expression gated on CD8+ TILs. C) Cytokine expression of Gata3+ and Gata3CD8+ TILs. Statistical analysis was performed using paired student t test. *p-value < 0.05, *** p-value < 0.001. D) Targeted deletion of Gata3 using CRISPR/Cas9 genome editing. Naïve CD8+ T cells were sorted from pmel transgenic mice, infected with control or Gata3 lentivirus and activated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (Methods and Resources). Representative qPCR results showing Gata3 mRNA level in control versus Gata3 lentivirus targeted CD8+ T cells. E) 1 × 106 CRISPR/Cas9-targeted cells were transferred to WT mice (n=5/group) bearing B16F10 melanoma tumors (day 5 post tumor grafting). Mean tumor growth is shown. Data are representative of 3 independent experiments. Statistical analysis was performed using linear regression. **p-value < 0.01. F and G) TILs were isolated on day 21 after tumor cell injection and analyzed for Tim-3 and PD-1 expression (F) and cytokine production (G) by flow cytometry.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat anti-PD-1 (clone: RMP1-30) Biolegend Cat#: 109109 Anti-Tim3 (clone: 5D12) Generated in house N/A Rat anti-IL-2 (clone: JES6-5H4) Biolegend Cat#: 503807 Anti-TNF-α (clone: MP6-XT22), eBioscience Cat#: 117321 Anti-IFN-γ (clone: XMG-1.2) Biolegend Cat# 505829 Mouse anti-Granzyme B (clone: GB11) Biolegend Cat#: 515405 Rat anti-CD8 (clone: 53–6.7) Biolegend Cat# 100731 Chemicals, Peptides, and Recombinant Proteins Zinpyr-1 Santa Cruz Cat#: sc-213182 Fixable viability dye eFluor506 eBioscience Cat#: 65-0866 Gp100 Genscript Cat#: RP20344 Critical Commercial Assays High Sensitivity DNA Kit (Bioanalyzer) Agilent Cat#: 5067-4626 Qubit dsDNA, High Sensitivity 500rxn Thermo Fisher Scientific Cat#: {"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}} Q32854 Nextera XT Sample Preparation Kit Illumina Cat#: FC-131-1096 NextSeq 500 high output kit V2, 75 cycles Illumina Cat#: FC-404-2005 Deposited Data Data files for CD8+ populations, Microarray This paper need to get accession number Data files for bulk RNA sequencing This paper need to get accession number Data files for single-cell RNA sequencing This paper need to get accession number LCMV exhaustion signature ( Doering et al., 2012 ) {"type":"entrez-geo","attrs":{"text":"GSE41867","term_id":"41867"}} GSE41867 CD8 + Ly49 + Treg signature ( Kim et al., 2015 ) {"type":"entrez-geo","attrs":{"text":"GSE73015","term_id":"73015"}} GSE73015 Experimental Models: Cell Lines MC38-OVA Mark Smyth N/A CT26 ATCC Cat#: CRL-2638 B16-F10 ATCC Cat#: CRL-6475 Experimental Models: Organisms/Strains Balb/c Jackson Laboratory Cat#: 000651 C57BL/6 Jackson Laboratory Cat#: 000664 PMEL Jackson Laboratory Cat#: 005023 OTI Jackson Laboratory Cat#: 003831 MT −/− (backcrossed to C57BL/6 in house) Jackson Laboratory Cat#: 002211 Recombinant DNA SMARTER TSO (with LNA, 10 µM)) Exiqon 5’- AAGCAGTGGTATC AACGCAGAGTACr GrG+G-3’ PCR oligonucleotide primer (10 µM) IDT 5’- AAGCAGTGGTATC AACGCAGAGT-3 Reverse Transcription DNA oligonucleotide primer (RNase-free, 100 µM) IDT 5- AAGCAGTGGTATC AACGCAGAGTACT (30)VN-3 Sequence-Based Reagents Gata3 CRISPR guide sequence Designed in house 5’ - GGTATCCTCCGAC CCACCACG Software and Algorithms GenePattern ( Reich et al., 2006 ) http://software.broadinstitute.org/cancer/software/genepattern/ COMBAT ( Johnson et al., 2007 ) http://www.bu.edu/jlab/wp-assets/ComBat/Download.html Bowtie ( Langmead et al., 2009 ) http://bowtie-bio.sourceforge.net/index.shtml RSEM ( Li and Dewey, 2011 ) http://deweylab.github.io/RSEM/ XL-mHG ( Wagner, 2015 ) https://github.com/flocompbio/xlmhg Other Open in a separate window KEY RESOURCES TABLE Distinct gene modules for T cell dysfunction and activation can be uncoupled.
Techniques: Binding Assay, Isolation, Expressing, Cell Function Assay, Flow Cytometry, CRISPR, Transgenic Assay, Infection, Injection
